Effect of Bacteriocin Production by Streptococcus mutans on

INFECTION AND IMMUNITY, Mar. 1979, p. 571-576 0019-9567/79/03-0571/00$02.00/00 Vol. 23, No. 3 Effect of Bacteriocin Production by Streptococcus mutans on the Plaque of Gnotobiotic Rats A. H. ROGERS,1 J. S. VAN DER HOEVEN,I* AND F. H. M. MIKXE Department of Preventive and Community Dentistry, University of Nijmegen, Nijmegen, The Netherlands,2 and Department of Oral Biology, University ofadelaide, Adelaide, South Australia' Received for publication 15 May 1978 The effect of bacteriocins produced by strains of Streptococcus mutans on the microbial composition of dental plaque was studied in gnotobiotic rats. In one set of tests, using S. mutans strains T2 and SW31, rats were simultaneously infected with the bacteriocinogenic parent (bac') and corresponding bac- mutants, and the relative proportions of each strain present in three different tooth sites were determined at various time intervals after inoculation. Some animals were monoinfected with bac' and bac- strains, and in vivo properties, such as plaque fermentation end products, amount of plaque formed, and cariogenicity, were measured. In another series of tests with strains T2 bac' and T2 bac-, animals were sequentially inoculated with a time interval of 1 week. On simultaneous inoculation, the bac- mutant, which was sensitive in vitro to the bacteriocin produced by the parent, was totally unable to establish in a plaque consisting of the bac' strain even when the challenging dose was increased 100-fold. In contrast, the bac' strain was successful not only in invading an established bac- plaque but also in eventually almost eliminating the mutant. In monoinfected animals, there were no significant differences in plaque-forming ability, fermentation end products, or cariogenicity between the two strains. Also, bacteriocin activity was detected in plaque harboring the bac' but not the bac- strain. The results not only indicate that bacteriocin activity can occur in vivo but also suggest that bacteriocinogeny might confer an ecological advantage upon a strain attempting to establish itself in an ecosystem. Previous work in this laboratory has suggested that bacteriocinogenic strains of Streptococcus mutans can prevent the establishment ofa strain of Actinomyces viscosus in the dental plaque of gnotobiotic rats, whereas the corresponding nonbacteriocinogenic (bac-) mutants cannot (8). These findings suggested that bacteriocins may play a role in modifying the plaque microflora. Recent in vitro experiments with a strain of S. mutans and a corresponding bac mutant have strengthened such a view (12). In studies of this type, it could be argued that the mutagen-induced bac mutants have been damaged in other respects, such as in the ability to colonize and to grow on the teeth, and that such changes were responsible for the observed results. Accordingly, in the present study, some animals were monoinfected with parent and bac- mutant strains, and various in vivo properties of the organisms were monitored. In other experiments, animals were simultaneously infected with the parent and mutant strains. In an additional experiment, one strain was allowed to establish for 1 week before challenge with the second strain. MATERIALS AND METHODS Microorganisms. The following strains of S. mutans were used: T2 (7); T2 bac-, a non-bacteriocinogenic mutant isolated after treatment of the parent strain with N-methyl-N'-nitro-N-nitroso-quanidine (Aldrich) (8); and SW31 (12) and its corresponding mutant, SW31 bac. These strains were usually grown in brain heart infusion broth (BBL) supplemented with 0.5% cysteine hydrochloride and 1% glucose; they were maintained by monthly subculture on blood agar. Both bac- mutants were made resistant to streptomycin to facilitate identification from mixed plaques and were designated T2 bac- S and SW31 bac- S. In a second set of experiments it was also necessary to use a streptomycin-resistant mutant of strain T2. Streptococcus sanguis NylOl (4), used as an indicator strain for bacteriocin production, was made resistant to erythromycin and designated NylOlE. Bacteriocin activity tests. The test strain was grown for 18 h at 370C in brain heart infusion broth; the culture was then centrifuged, the pelleted cells were stab-inoculated into Trypticase soy agar (8), and the plates were incubated for 48 h at 370C under an atmosphere of 85% N2, 10% H2, and 5% C02. Plates were then seeded with 4.5 ml of Trypticase soy agar containing only 0.75% agar and about 0.2 ml of an 571

572 ROGERS, VAN DER HOEVEN, AND MIKX overnight brain heart infusion broth culture of the indicator strain. After overnight aerobic incubation at 37CC, plates were examined for zones of inhibition. Where indicated below, plaque samples from four or five animals in treatment groups 1, 2, 4, and 5 were also tested for bacteriocin activity. This was done by transferring a sample of plaque directly to the surface of a Trypticase soy agar plate containing 200 jig of erythromycin per ml. Strain NylOlE was used as the indicator, and the overlay also contained 200 Mg of erythromycin per ml. After overnight aerobic incubation at 370C, plates were examined for zones of inhibition. The effectiveness of erythromycin in preventing growth of the plaque organism was demonstrated by streaking these samples on the surface of Trypticase soy-erythromycin agar. It should also be pointed out here that in vitro tests carried out on solid media had previously shown (8) that each of the two parent strains demonstrated bacteriocin-like activity against its corresponding bacmutant, the effect being much more marked in the case of strains T2 and T2 bac. Experimental procedures. Gnotobiotic Osborne- Mendel rats, 25 to 30 days old, were used. They were kept in Macrolon cages without bedding; diet 540 S (5) and drinking water were available ad libitum. Sixteen animals were used per group; in four of the six groups the rats were monoinfected, and in the other two they were inoculated simultaneously with two strains. The groups were as follows: 1, T2; 2, T2 bac- S; 3, T2 + T2 bac- S; 4, SW31; 5, SW31 bac- S; 6, SW31 + SW31 bac- S. Each inoculum consisted of 0.1 ml of an overnight culture of the appropriate organism in brain heart infusion broth containing 0.1% glucose and was delivered with a tuberculin syringe. To ensure that all animals received approximately the same dose of organisms in groups 3 and 6, where two strains were simultaneously inoculated, each strain was suspended in half its original volume and mixed in equal amounts before use. In this way, each animal received 2 x 10' to 4 x 108 colony-forming units of each strain on each of 2 consecutive inoculation days. On days 1, 6, 13, and 41 after the second of the two inoculations with S. mutans, separate samples were obtained from four rats of each of the six groups from the mesial surface and the central fissure of the lower left second molar and from an area between the first and second lower left molars. The samples were designated fissure, smooth surface, and approximal. Samples were removed with sterile dental probes, suspended in 100!d of 0.1 M histidine, and homogenized (8). In a subsequent experiment involving S. mutans strains T2 and T2 bac-, the two strains were not simultaneously inoculated, but instead one was allowed to establish for 1 week prior to challenge with the second strain. The groups, following sequentially from the six listed above, were as follows: 7, T2 + T2 bac- S; 8, T2 + T2 S; 9, T2 bac- S + T2; 10, T2 S + T2; 11, T2 + T2 bac S (x102); 12, T2 bac- S + T2 (x102). For the rats in groups 11 and 12, an inoculum 10Ox higher than the usual 1 x 108 to 4 x 108 was used. In all other respects the protocol for groups 7 to 12 was the same as described above for groups 1 to 6. However, in this second series, plaques were sampled on days 2, 6, 13, INFECT. IMMUN. and 29 after the second of the two inoculations with the challenging organism. In addition, fermentation end products and caries scores (see below) were not determined for animals in treatments 7 to 12. Bacteriological studies. From each dispersed sample, 0.1-ml amounts of the appropriate dilutions were spread on both Trypticase soy agar (BBL) and Trypticase soy agar containing 100Mig of streptomycin per ml. Colony counts were done after incubation for 48 h in an N2-H2-CO2 atmosphere. The samples from animals in groups 3 and 6 contained both parent (bac') and mutant (bac- S) strains, and differential counts could thus be made on the two media. In addition, appropriate Trypticase soy agar plates were subsequently seeded with the indicator NylO1, thereby affording another method of differentially counting the bac' and bac- strains. Plaque samples were also taken to ensure that the rats continued to harbor only the strain with which they were originally infected. Fermentation products and DNA in plaque samples. Dental plaque was removed quantitatively from the central fissures of three molars on one side of the lower jaw and pooled, giving one pooled sample per rat; as noted above, there were four rats per treatment group. Lactic and acetic acids were assayed isotachophoretically (2), and ethanol and DNA were determined as described previously (10). These tests were performed only on plaque from rats monoinfected with either strain T2 or T2 bac- S (groups 1 and 2). Caries scores and plaque development. Fissure caries and plaque development were scored according to Konig (2, 3). RESULTS Establishment of S. mutans parent and bac mutants in plaque after simultaneous inoculation. Although the mutant strain T2 bac- S was established in early (1-day-old) plaque equal to the parent strain, its proportion in relation to the parent strain steadily declined in time (Fig. 1). The same trend occurred with strain SW31 bac- S and its parent, although the initial establishment of this mutant was rather poor (Fig. 2). Differential counting of the strains by bacteriocin production gave essentially the same pattern as described above. Bacteriocin activity in plaque samples. The results for S. mutans T2 parent and mutant strains are listed in Table 1, from which it would appear that the plaques from animals monoinfected with the bac' strain produced zones of inhibition against S. sanguis NylOlE, whereas those harboring the bac mutant did not. Direct measurement showed no significant difference in ph between areas within inhibition zones and comparable areas surrounding the bac- plaques. Moreover, the inhibitory activity from the T2 plaque was not destroyed by trypsin treatment, thus confirming previous in vitro results (7). No inhibitory activity could be detected in plaques formed by either SW31 parent or mutant strains.

VOL. 23, 1979 BACTERIOCIN ACTIVITY IN THE PLAQUE OF RATS 573 % T2 Bac-S Simultaneous inoculation of & mutons T2 and T2 Bac- S 0 fissure - - - smooth surface.approximally 1.1 A 70-60- 50-40- 30-20- 10-9 0 -- I - I - - I l 2 4 10 20 30 40 day FIG. 1. Percentage of S. mutans T2 bac- S in fissure, smooth surface, and approximal plaque in gnotobiotic rats following simultaneous inoculation of the rats with S. mutans T2 and T2 bac S. _- I Bac- S - smooth fissure surface....approximally I 2, 10, 20 30 40 day _ FIG. 2. Percentage of S. mutans SW31 bac- S in fissure, smooth surface, and approximal plaque in gnotobiotic rats following simultaneous inoculation of the rats with S. mutans SW31 and SW31 bac- S. Fermentation end products in plaques. There appeared to be no significant difference between strains T2 and T2 bac- either in terms of the amount of plaque formed, as measured by amounts of bacterial DNA present, or with respect to fermentation end products present (Table 2). Caries and plaque scores. With regard to both strains T2 and SW31, there appeared to be no significant difference between the parent and the corresponding bac- mutant in terms of the amounts of plaque formed and fissure caries produced (Table 3). None of these strains induced smooth-surface caries in the gnotobiotic rats. The effect of sequential inoculation on the establishment of S. mutans strains T2 TABLE 1. Demonstration of bacteriocin activity in plaque samples from germfree rats infected with S. mutans S. mutans strain Characterization of plaque samples T2 T2 T2 + T2 SW31 bac- S bac- S Growth in presence of - - - - erythromycin (200 ug/ ml) Inhibition against S. san- +a _ + guis NylOlE aactivity insensitive to trypsin (1 mg/ml). and T2 bac-. When the mutant was used to challenge a 1-week-old plaque formed by the parent strain (Table 4, group 7), it did not establish even in early (2-day-old) plaque. This was

574 ROGERS, VAN DER HOEVEN, AND MIKX so even when the challenging dose was increased 100-fold (group 11). In direct contrast, when the parent was used to challenge a plaque formed by the mutant, it quickly became established and eventually outnumbered the mutant (group 9). This effect was even more marked when the challenging dose was increased 100-fold; the mutant was almost completely eliminated after 6 days (group 12). In general, these results were the same for all three sampling sites, but some differences were TABLE 2. Lactic and acetic acids, ethanol, and DNA in the plaque ofgnotobiotic rats monoinfected with S. mutansa Substance S. mutans strain Lactic Acetic b DNA acidb acidb Ethanol' 4 T2 70 ± 20 200 ± 70 160 ±10 0.17 ± 0.66 T2 bac- S 100 ± 50 190 ± 100 170 ± 130 0.22 ± 0.09 a Four rats per treatment group. Expressed as nanomoles per microgram of DNA; mean and standard deviation. TABLE 3. Caries and plaque scores in gnotobiotic rats monoinfected with S. mutansa Fissure lesions Plaque S. mutans strain Pau T B scores T2 5.9 ± 1.2 4.6 + 2.0 1.6 + 0.7 T2 bac-s 5.9 + 1.2 5.0 + 1.6 1.5 ± 0.5 SW31 7.2 + 1.1 6.6 + 1.3 1.7 ± 0.5 SW31 bac- S 6.8 ± 0.9 6.2 + 0.9 1.5 + 0.5 a Sixteen rats per treatment group. TABLE 4. apparent. In particular, whereas the bac' challenger almost entirely eliminated the previously established bac strain from both fissure and smooth-surface plaques, in the approximal site the two strains were found in about equal proportions (group 9, Fig. 3). %T2 100-80- 60-40- 20- *I fissure I S. mutans T2 Bac plaque challenged by the parent (Bac ) strain smooth surface a~~~~~~pproximal INFECT. IMMUN. 2 10 20 30 day FIG. 3. Percentage of S. mutans T2 in fissure, smooth surface, and approximal plaque in gnotobiotic rats inoculated with S. mutans T2 bac- S and 1 week later with S. mutans T2. Establishment of S. mutans strains T2 and T2 bac- S in the plaque ofgnotobiotic rats after sequential inoculation Presence of challenging strain in group: Days post. Sample' T/2 8(2/2 9(2bc5 challenge 7 (T2/T2 (T/ 9 M bac SI 10 (T2 S.T2) 11 (T2/T2 bac 12 (T2 bac SI/ bac S)C S) T2) / S [X102]) T2 [x102]) 2 F 0 0 20 0 0 19 S 0 0 3.0 0 0 31 A 0 0 60 9.0 0 58 6 F 0 0 30 6.0 0 100 S 0 0 100 24 1.0 99 A 0 0 60 17 <0.1 100 13 F 0.1 0 91 1.0 <0.1 95 S 0 0 99 0 0 99 A 0 0 78 0 0 94 29 F 0 0 95 0.5 <0.1 >99 S 0 0 100 0 0 >99 A 0 0 52 0 0 97 a F, Fissure; S, smooth surface; A, approximal. b Expressed as median percentage of total of both strains. Parentheses give established plaque/challenge strains.

VOL. 23, 1979 DISCUSSION When simultaneously inoculated, S. mutans strains T2 and T2 bac- colonized equally well on the teeth of gnotobiotic rats but, with the passage of time, the relative proportion of the bac- mutant decreased to low levels in all three tooth sites tested. Much the same is also evident from the results for strain SW31, although the bac- mutant initially appeared to colonize less well than the parent strain. The validity of the observations was strengthened by the fact that streptomycin-resistant strains colonized similar to nonresistant parental strains (Table 4, groups 8 and 10). On sequential inoculation, where the parent T2 strain was allowed to stabilize itself for a week on the tooth surface before challenge with the bac- mutant, the latter was totally unable to establish even when the challenging dose was increased 100-fold. However, the parent (bac') was successful not only in invading an established plaque formed by the bac- mutant but also in eventually almost eliminating the mutant. The fact that such a phenomenon was not so evident in approximal plaques indicates that the areas sampled represent different ecological niches. It is not likely that the present findings could be ascribed to marked differences in biological properties between the parent and mutant strains, since their comparative plaque-forming capacities as judged from plaque scores and quantitative sampling of fissure plaque, plaque fermentation products, and ultimate ability to produce dental caries were not significantly different. In passing it might be noted that the finding of a mixed fermentation in these plaques confirms previous studies with another strain of S. mutans (9). Antibacterial activity was demonstrated in natural plaque formed by S. mutans T2 but not from plaque formed by its bac- mutant. The activity was not impaired by trypsin, and previous work (7) has shown that in vitro T2 bacteriocin activity is also insensitive to this enzyme. Bacteriocin activity from S. mutans SW31 in vitro-grown plaque has recently been demonstrated (12), but the present study failed to reveal such activity in natural plaque formed by this strain. This is perhaps not surprising, since the in vitro bacteriocin activity of strain SW31 is much weaker than that of strain T2. Moreover, comparison between in vitro and natural plaques is not necessarily valid. At least in the case of T2, the above in vitro finding has now been confirmed in vivo, and, in addition, it substantiates a previous study (8) wherein a strain of A. viscosus was kept at low levels in the presence of bac' parent strains of S. mutans but not by BACTERIOCIN ACTIVITY IN THE PLAQUE OF RATS 575 the corresponding bac- mutants in the plaque of gnotobiotic rats. Hardy (1), in an extensive review of the subject, concluded that the role of bacteriocins in influencing microbial ecology in natural environments is equivocal. Recently, however, it was reported that not only is colicin V produced in vivo by certain strains of E. coli but that it may also act as a virulence factor in some strains (6). Furthermore, following their demonstration of the role of bacteriocins in modifying in vitro plaque micro-ecology, Weerkamp and his coworkers (12) suggested that this might also be the case in vivo. The current study and a previous one (8) provide some of the necessary evidence to support this contention. The present study indicated bacteriocin activity in a simple ecosystem consisting of only two organisms; it also indicated the way in which that bacteriocinogeny was a definite ecological advantage to a strain attempting to establish itself in a simple ecosystem. This hypothesis should be tested in a more complicated natural situation, such as that which exists in specificpathogen-free or conventional rats. Such studies are now being undertaken. ACKNOWLEDGMENTS We are grateful for the skilled technical assistance of Gabrielle Raaymaakers and Jan Lukassen. One of us (A.H.R.) was supported by a grant from The Netherlands Organization for the Advancement of Pure Scientific Research. LITERATURE CITED 1. Hardy, K. G. 1975. Colicinogeny and related phenomena. Bacteriol. Rev. 39:464-515. 2. Konig, K. G. 1959. Dental caries and plaque accumulation in rats treated with stannous fluoride and penicillin. Helv. Odont. Acta 3:39-44. 3. Kolig, K. G. 1966. Moglichkeiten der Kariesprophylaxe beim Menschen und ihre Untersuchung im Ku-dristigen Rattenexperiment. Hans Huber, BernL 4. Mikx, F. H. M., J. S. van der Hoeven, A. J. M. Plasschaert, and K. G. Konig. 1975. Effect ofactinomyces viscosus on the establishment and symbiosis of Streptococcus mutans and Streptococcus sanguis in SPF rats on different sucrose diets. Carries Res. 9:1-20. 5. Mikx, F. H. M., J. S. van der Hoeven, A. J. M. Plaswchaert, and K. G. Konig. 1976. The establishment and symbiosis of Actinomyces viwcosus, Streptococcus sanguis and Streptococcus mutans in germ-free Osborne-Mendel rats. Carries Res. 10:123-132. 6. Ozanne, G., L. G. Mathieu, and J. P. Baril. 1977. Production of colicin V in vitro and in vivo and observations on its effect in experimental animals. Infect. Immun. 17:497-503. 7. Rogers, A. H. 1976. Bacteriocinogeny and the properties of some bacteriocins of Streptococcus mutans. Arch. Oral Biol. 21:99-104. 8. Rogers, A. H., J. S. van der Hoeven, and F. H. M. Mikx. 1978. Inhibition of Actinomyces viscosus by bacteriocin-producing strains of Streptococcus mutans in the dental plaque of gnotobiotic rats. Arch. Oral Biol. 23:477-483. 9. van der Hoeven, J. S. 1976. Carbohydrate metabolism

576 ROGERS, VAN DER HOEVEN, AND MIKX of Streptococcus mutans in dental plaque in gnotobiotic rats. Arch. Oral Biol. 21:431-433. 10. van der Hoeven, J. S., H. C. M. Franken, P. J. M. Camp, and C. W. Dellebarre. 1978. The analysis of bacterial fermentation products by isotachophoresis. Apple. Environ. Microbiol. 35:17-23. 11. van derrioeven, J. S., A. I. Toorop, and F. H. M. INFECT. IMMUN. Mikx. 1978. Symbiotic relationship of Veillonella alcalescens and Streptococcus mutans in dental plaque in gnotobiotic rats. Caries Res. 12:142-148. 12. Weerkamp, A., L. Bongaerts-Larik, and G. D. Vogels. 1977. Bacteriocins as factors in the in vitro interaction between oral streptococci in plaque. Infect. Immun. 16:773-780.